基于叠氮基-氰基点击化学的苯硼酸亲和硅胶的制备及其在糖蛋白/糖肽选择性富集中的应用

硼亲和色谱法在糖肽/糖蛋白选择性富集中的应用趋于成熟。硼酸亲和材料的选择性，生物相容性，制备过程是否简便均是开发新型苯硼酸功能化材料需要考虑的问题。该研究立足硼酸亲和材料开发的关键问题，设计并开发了一种新型苯硼酸亲和硅胶（TCNBA）。该材料采用基于叠氮基-氰基的无铜催化点击化学方法进行合成，生物相容性好，制备方法简便。红外光谱和X射线光电子能谱图表征结果证明材料合成成功。TCNBA的糖肽富集选择性利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行评价，结果表明，TCNBA能够分别从辣根过氧化物酶（HRP）和免疫球蛋白G（IgG）酶解液中鉴定出13个和11个糖肽；以HRP和牛血清白蛋白（BSA）酶解液混合物（物质的量比1：10）作为研究对象，富集后能够鉴定出5个糖肽。TCNBA的糖蛋白富集选择性利用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法（SDS-PAGE）进行评价，以HRP、IgG、核糖核酸酶B（RNaseB）作为考察对象，结果表明，TCNBA对糖蛋白具有较好的富集选择性。以实际样品人血清为测试对象验证TCNBA在实际生物样品中的应用价值。结果显示，富集后非糖蛋白得到较大程度去除，糖蛋白得以富集。所制备的材料和方法具有大规模实际蛋白质样品分离处理的应用前景。     

一种弱阳离子交换材料的亲水模式糖肽富集新方法

亲水作用色谱(HILIC)材料在糖肽的富集与分离中得到越来越广泛的应用. 针对目前HILIC材料糖肽选择性不足的缺点, 发展新的糖肽富集方法十分必要. 本工作发展了一种商品化的弱阳离子交换材料(WCX)在亲水模式下对糖肽的富集方法. 首先考察了微固相萃取(SPE)模式下WCX上多肽保留的机理. 结果显示, WCX上糖肽的保留同时受乙腈含量和pH的影响. 将WCX用于牛胎球蛋白(fetuin)胰蛋白酶酶解液中糖肽的富集, 获得了39条糖肽信号. 当fetuin与牛血清白蛋白(BSA)酶解液以物质的量比1:5混合时, 富集到26条糖肽信号. 富集人免疫球蛋白(IgG)胰蛋白酶酶解液时获得25条糖肽信号, 1:5时获得23条糖肽信号. 实验说明WCX对唾液酸化和非唾液酸化糖肽的富集均有高选择性. 该方法拓展了WCX的应用范围, 丰富了HILIC材料的种类.     

新型四肽亲水材料用于糖肽的高效富集

本研究采用原子转移自由聚合(Atom-transfer radical-polymerization, ATRP)法合成了一种新型四肽亲水作用色谱材料 (Poly-DAPD),用于糖肽的选择性富集.通过氮气吸脱附、热重分析和X射线光电子能谱等技术进行表征,结果表明,四肽已成功接枝到硅球上.固相萃取富集实验表明,合成的亲水材料对牛胎球蛋白(Fetuin)糖肽富集选择性高;与商品化ZIC-HILIC材料相比,Poly-DAPD材料富集掺有5摩尔倍数牛血清蛋白(BSA)的Fetuin样品时,在获得的糖肽数目及抗干扰性能方面都更具优势.此Poly-DAPD材料可进一步用于不同糖蛋白的糖基化分析研究.     

基于纳米粒子的糖蛋白/糖肽分离富集方法

蛋白质糖基化是一种重要的蛋白质翻译后修饰方式,糖基化对蛋白质的结构和功能有着非常重要的影响。在血清或者组织提取液中,一些低浓度的糖蛋白/糖肽是具有高度临床灵敏性和特异性的生物标记物,这些生物分子可能对疾病发生机理探讨、疾病标记物发现及蛋白类新药开发提供重要信息。由于糖蛋白/糖肽的丰度低,从复杂的生物样品中高选择性富集糖蛋白/糖肽一直是糖蛋白组学的难点和重点。纳米结构的材料因其大比表面积、丰富的活性亲和位点和特殊结构,已经广泛应用于糖蛋白/糖肽的分离富集中。本文对基于金、SiO₂、TiO₂、Fe₃O₄、金刚石和聚合物纳米粒子为载体的糖蛋白/糖肽分离富集方法的研究进展作了简要概述,并且阐明了糖蛋白/糖肽分离富集方法所面临的挑战,最后,对其未来发展方向做了展望。     

小尺度4-巯基苯硼酸修饰纳米金粒子用于大鼠肝脏糖蛋白选择性富集和质谱鉴定研究

报道了利用硼氢化钠还原一步法合成小尺度巯基苯硼酸修饰的纳米金材料（尺寸约为3 nm）,合成方法简便快捷.利用纳米金表面的硼酸基团与糖蛋白/糖肽中顺式邻二羟基的酯化反应,成功实现了糖肽和糖蛋白的选择性富集.利用该材料对糖蛋白HRP酶解产物进行富集,经富集后糖肽质谱峰的信号强度提高近百倍;将该材料用于大鼠肝脏中糖蛋白的富集...     

钴酞菁功能化聚合物整体柱的制备及用于转铁蛋白糖肽的富集

建立了一种新型的聚合物整体柱制备方法,将四羧基钴酞菁（CoPcTc）引入聚（甲基丙烯酸缩水甘油酯-乙二醇二甲基丙烯酸酯）（Poly（GMA-EDMA））整体柱,用于转铁蛋白糖肽的富集。由于四羧基钴酞菁和糖肽之间的氢键和金属配位作用,制备的整体材料在用于糖肽富集时具有高效性和选择性。转铁蛋白经过功能化整体柱富集后,通过电喷雾四极杆飞行时间质谱分析,共捕获到17条糖肽。当转铁蛋白浓度降至8.8×10^-10mol/L时,仍然可以得到3条糖肽,说明合成的聚合物整体材料对微量蛋白样品分析具有很高的应用潜力。     

共价有机骨架功能材料及其在糖肽选择性富集中的应用

蛋白质糖基化是生物体中最重要的翻译后修饰手段之一,糖蛋白/糖肽的有效分离和富集成为目前糖蛋白组学研究的首要问题.对于复杂的生物样本,糖蛋白的数量较少,酶解后大量高丰度非糖基化修饰肽的存在,使得低丰度糖肽的检测更加困难.因此,需要一些手段来有效地富集糖肽以提高其检测丰度,发展高选择性的糖肽富集材料及方法就成为在分子水平上...     

智能聚合物基材料富集磷酸化肽和糖肽的研究进展

翻译后修饰是蛋白质组学研究的前沿和重点,它不仅调节着蛋白质的折叠、状态、活性、定位以及蛋白质间的相互作用,也能帮助科学家更全面地了解生物体的生命过程,为疾病的预测、诊断和治疗提供更加强大的支撑和依据。翻译后修饰产物(例如磷酸化肽和糖肽)丰度很低,且存在着强烈的背景干扰,很难直接用质谱进行分析,因此迫切需要开发高效的富集材料和技术来选择性富集翻译后修饰产物。近年来,智能聚合物基材料通过外部物理、化学或生物刺激可逆地改变其结构和功能,实现对磷酸化肽和糖肽高度可控的吸附和脱附,进而衍生开发出一系列新颖的富集方法,极大地吸引研究者们的兴趣。一方面,智能聚合物基材料的响应变化包括材料疏水性的增加或减少、形状和形貌的改变、表面电荷的重新分布以及亲和配体的暴露或隐藏等特性。这些特性使得目标物和智能聚合物基材料之间的亲和力可以通过简单改变外部条件(如温度、pH值、溶剂极性和生物分子等)实现更可控和更智能的精细调节。另一方面,智能聚合物基材料为集成功能模块提供了便捷的可扩展平台,例如特定的识别组件,显著提高了目标物质的分离选择性。智能聚合物基材料在分离方面展现出巨大的潜力,这为蛋白质翻译后修饰产物的分析和研究带来了希望。围绕上述主题,该文依据Web of Science近20年来近50篇代表性文献,概述了智能聚合物基材料在磷酸化肽和糖肽分离及富集中的发展方向。     

硅胶修饰-表面分子印迹牛血红蛋白及其识别性能的研究

选用马来酸酐修饰的硅胶作为载体,丙烯酰胺为功能单体,N,N′-亚甲基双丙烯酰胺为交联剂,牛血红蛋白为模板分子,采用氧化还原悬浮聚合法,合成了具有选择性识别的牛血红蛋白分子印迹聚合物.并用红外光谱(IR)、扫描电子显微镜(SEM)和元素分析对聚合物进行了表征,结果表明载体表面成功地接枝了分子印迹聚合物薄层.同时选择性吸附实验表明分子印迹聚合物具有良好的识别性能,能实现水溶液中牛血红蛋白的富集.     

修饰硅胶表面印迹牛血红蛋白及其识别性能的研究

本文选用马来酸酐修饰后的硅胶作为载体,丙烯酰胺为功能单体,N,N’-亚甲基双丙烯酰胺为交联剂,牛血红蛋白为模板分子,采用氧化还原悬浮聚合法,合成了具有选择性识别的牛血红蛋白分子印迹聚合物。并用红外光谱(IR)、扫描电子显微镜(SEM)对聚合物进行了表征,结果表明载体表面成功接枝了分子印迹聚合物薄层。同时,选择性吸附实验表明分子印迹聚合物的具有良好的识别性能,能成功的实现水溶液中牛血红蛋白的富集。     

智能响应材料在磷酸化肽和糖肽富集中的应用

蛋白质的磷酸化和糖基化作为研究最广泛的两种翻译后修饰(PTMs),在疾病的早期无创诊断、预后和治疗评估中表现出越来越大的潜力。蛋白质的异常磷酸化和糖基化经常被用于临床蛋白质组学研究和疾病相关生物标志物的发现。目前已有多种材料被开发用于磷酸化肽和糖肽的富集研究,其中,智能响应材料由于具有独特的响应特性,已被陆续报道用于磷酸化肽和糖肽的富集。智能响应材料可对外界刺激做出响应,发生结构和性质上的变化,将光、电、热、机械等信号转化为生物化学信号。响应分子是决定智能响应材料响应特性的先决条件,它们在不同刺激条件下(如温度、pH、光、机械应力、电磁场等)的可逆异构化将导致材料的宏观物理和化学性质的动态变化。与传统材料相比,智能响应材料可以可逆地“打开”和“关闭”,具有更好的可调控性。由于引起智能材料响应的刺激信号对其性能具有重要的影响,综述根据施加的刺激种类对智能响应材料进行分类,具体分为外源性响应材料和内源性响应材料,且分别总结了外源性响应材料、内源性响应材料以及内外源共同响应材料在磷酸化肽和糖肽富集方面的工作。此外,综述对智能响应材料在磷酸化肽和糖肽富集方面的发展前景进行了展望,并且提出了智能响应材料在其他蛋白质翻译后修饰方面的应用中存在的挑战。     

氧化石墨烯固定化凝集素的制备及在糖蛋白/糖肽富集中的应用

生物体内蛋白质的糖基化修饰调控着细胞识别、细胞黏附和迁移以及免疫应答等多种生理过程,并与多种人类重大疾病的发生、发展密切相关。因此对蛋白质糖基化修饰的鉴定,不仅能够为生物学机理研究提供重要信息,对疾病诊断标志物和治疗靶标的发现也至关重要。然而在复杂生物体系中,大多数糖蛋白为低丰度蛋白质,其含量与现有质谱仪器的检测灵敏度之间存在较大差距,所以对含有不同糖型结构的糖蛋白进行全面/高效的富集,是实现高灵敏度糖蛋白鉴定的必由之路。凝集素富集作为一种有效的糖蛋白富集方法,已在糖蛋白质组学研究中得到了广泛的应用。针对现有凝集素功能化材料存在负载量偏低以及富集效率有限等问题,我们制备了两种以氧化石墨烯(GO)为载体的新型固定化凝集素,利用GO比表面积大,功能基团含量高,分散性、化学稳定性好等特点,实现了高负载量的凝集素固定(GO-ConA 2.073 mg/mg, RSD=1.0%; GO-WGA 1.908 mg/mg, RSD=0.14%)。同时考察了材料的可重复使用性与稳定性:每隔3天测一次同一GO-lectin材料对对应糖蛋白的富集效果,可以看出材料合成两周内富集效果都>200 μg/mg。将该GO-lectin成功应用于糖蛋白、糖肽的选择性富集,在糖蛋白质组学研究中体现出良好的应用潜力。     

Facile preparation of hydrophilic glutathione modified magnetic nanomaterials for specific enrichment of glycopeptides

Glutathione modified magnetic nanoparticles (Fe₃O₄@Au-GSH) were synthesized through a simple process and exploited to enrich glycopeptides from complex samples.     

Facile preparation of hydrophilic glutathione modified magnetic nanomaterials for specific enrichment of glycopeptides

Investigations of glycosylated proteins or peptides and their related biological pathways provide new possibilities for illuminating the physiological and pathological mechanisms of glycosylation modification. However, open-ended and in-depth analysis of glycoproteomics is usually subjected to the low-abundance of glycopeptides, heterogeneous glycans, and a variety of interference molecules. In order to alleviate the influence of these obstacles, effective preconcentration of glycopeptides are indispensable. Here, we employed a hydrophilic interaction liquid chromatography (HILIC)-based method to universally capture glycopeptides. Glutathione modified magnetic nanoparticles (Fe₃O₄@Au-GSH) were synthesized through a simple process and exploited to enrich glycopeptides from complex samples. The prepared materials showed excellent ability to trap glycopeptides from standard glycoproteins digests, low detection limit (10 fmol/μL), and good selectivity (HRP:BSA = 1:100). These results indicated that glutathione-based magnetic nanoparticles synthesized in this work had great potential for glycopeptides enrichment.     

两性离子双功能化超亲水金属有机框架纳米复合材料的制备及其用于糖肽的选择性富集

蛋白糖基化是生物体中普遍发生且重要的生物学过程,其参与多种分子生物学的功能和途径,是临床诊断重要的生物标志物.但是,糖肽因其丰度低、离子化效率低、糖链异质性等难点,使糖蛋白分析一直面临巨大的挑战.因此,研究合成了一种新型的两性离子双功能化纳米金(AuGC)修饰的超亲水性沸石咪唑骨架(ZIF-8)纳米复合材料(AuGC/...     

Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic glycopeptides

Protein glycosylation has a significant medical importance as changes in glycosylation patterns have been associated with a number of diseases. Therefore, monitoring potential changes in glycan profiles, and the microheterogeneities associated with glycosylation sites, are becoming increasingly important in the search for disease biomarkers. Highly efficient separations and sensitive methods must be developed to effectively monitor changes in the glycoproteome. These methods must not discriminate against hydrophobic or hydrophilic analytes. The use of activated graphitized carbon as a desalting media and a stationary phase for the purification and the separation of glycans, and as a stationary phase for the separation of small glycopeptides, has previously been reported. Here, we describe the use of activated graphitized carbon as a stationary phase for the separation of hydrophilic tryptic glycopeptides, employing a chip‐based liquid chromatographic (LC) system. The capabilities of both activated graphitized carbon and C₁₈ LC chips for the characterization of the glycopeptides appeared to be comparable. Adequate retention time reproducibility was achieved for both packing types in the chip format. However, hydrophilic glycopeptides were preferentially retained on the activated graphitized carbon chip, thus allowing the identification of hydrophilic glycopeptides which were not effectively retained on C₁₈ chips. On the other hand, hydrophobic glycopeptides were better retained on C₁₈ chips. Characterization of the glycosylation sites of glycoproteins possessing both hydrophilic and hydrophobic glycopeptides is comprehensively achieved using both media. This is feasible considering the limited amount of sample required per analysis (<1 pmol). The performance of both media also appeared comparable when analyzing a four‐protein mixture. Similar sequence coverage and MASCOT ion scores were observed for all proteins when using either stationary phase. Copyright © 2009 John Wiley & Sons, Ltd.     

Improvement of mass spectrometry analysis of glycoproteins by MALDI-MS using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid

Protein glycosylation analysis is important for elucidating protein function and molecular mechanisms in various biological processes. We previously developed a glycan analysis method using a 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid liquid matrix (3-AQ/CHCA LM) and applied it to the quantitative glycan profiling of glycoproteins. However, information concerning glycosylation sites is lost; glycopeptide analysis is therefore required to identify the glycosylation sites in glycoproteins. Human epidermal growth factor receptor 2 (HER2) is a glycoprotein that plays a role in the regulation of cell proliferation, differentiation, and migration. Several reports have described the structure of HER2, but the structures of N-glycans attached to this protein remain to be fully elucidated. In this study, 3-AQ/CHCA LM was applied to tryptic digests of HER2 to reveal its N-glycosylation state and to evaluate the utility of this LM in characterizing glycopeptides. Peptide sequence coverage was considerably improved compared to analysis of HER2 using α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid. Most of the peaks observed using only this LM were localized at the inner or outer regions of sample spots. Furthermore, five of the peptide peaks that were enriched within the inner region were confirmed to be glycosylated by MS/MS analysis. Three glycosylation sites were identified and their glycan structures were elucidated. The reduction in sample complexity by on-target separation allowed for higher sequence coverage, resulting in effective detection and characterization of glycopeptides. In conclusion, these results demonstrate that MS-based glycoprotein analysis using 3-AQ/CHCA is an effective method to identify glycosylation sites in proteins and to elucidate the glycan structures of glycoproteins in complex samples.     

Label-free quantitation: A new glycoproteomics approach

We demonstrate herein a method for quantifying glycosylation changes on glycoproteins. This novel method uses MS data of characterized glycopeptides to analyze glycosylation profiles, and several quality control tests were done to demonstrate that the method is reproducible, robust, applicable to different types of glycoproteins, and tolerant of instrumental variability during ionization of the analytes. This method is unique in that it is the first label-free quantitative method specifically designed for glycopeptide analysis. It can be used to monitor changes in glycosylation in a glycosylation site-specific manner on a single glycoprotein, or it can be used to quantify glycosylation in a glycoprotein mixture. During mixture analysis, the method can discriminate between changes in glycosylation of a given protein, and changes in the glycoprotein’s concentration in the mixture. This method is useful for quantitative analyses in biochemical studies of glycoproteins, where changes in glycosylation composition can be linked to functional differences; it could also be implemented in the pharmaceutical industry, where glycosylation profiles of glycoprotein-based therapeutics must be quantified. Finally, quantification of glycopeptides is an important aspect of glycopeptide-based biomarker discovery, and our quantitative approach could be a valuable asset to this field as well, provided the compositions of the glycopeptides to be quantified are identifiable using other methods.     

Glycosylation analysis of interleukin-23 receptor: elucidation of glycosylation sites and characterization of attached glycan structures

Interleukin-23 (IL-23) is a heterodimeric cytokine, a central factor in chronic/autoimmune inflammation. It signals through a heterodimeric receptor consisting of IL-23r, which is heavily glycosylated. The structural characterization of IL-23r has not been reported. In this work, glycosylation profiles of soluble recombinant human IL-23r (rhIL-23r) were established using mass spectrometry (MS), which included defining glycosylation sites, degree of glycosylation occupancy of each site and structure of attached oligosaccharides. Specifically, precursor ion scan of oxonium ion protonated N-acetylglucosamine (GlcNAc(+)) (m/z 204) was performed using a triple quadrupole MS instrument to locate the retention time of glycopeptides. Both the glycopeptides and their corresponding deglycosylated forms in each collected HPLC fraction were studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (LTQ-Orbitrap) for glycosylation site profiling. The attached glycan structures were elucidated by collision-induced dissociation (CID) fragmentation of target glycopeptides in combination with accurate mass measurement. Eight glycosylation sites were identified on IL-23r (Asn24, Asn209, Asn239, Asn157, Asn118, Asn250, Asn58 and Asn6). Most of the glycosylation sites were > 95% occupied except Asn250 and Asn6. Those two sites were 88% and 45% occupied by estimation from trypsin digestion and were 55% and 42% occupied from LysC digestion. Multiple glycoforms were observed in IL-23r. Most of them were bi-, tri- or tetra-antennary complex type structures with fucose and sialic acid. High mannose and hybrid type glycans were only observed on Asn157. The structural characterization on IL-23r glycosylation provides useful information for better understanding of the biological function of IL-23r.